Sample Set Annotation Tool: Genome-wide profiling of whole blood from patients with defects in Toll-like receptors and IL-1Rs signaling-GSE25742

Study Design
Assay
Sample Info
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Samples
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Analysis

Purpose

The objective of this study is to: 1) Characterize the innate immune responsiveness of patients with inborn errors in Toll-IL1 receptor signaling pathway (IRAK4, MyD88 deficiencies) compared to healthy subjects, through the analysis of blood leukocytes' transcriptional profiles after stimulation with ligands for the whole set of Toll-like receptors and IL-1Rs plus whole bacteria. 2) Understand the redundancies in TLR pathway in humans. 3) Explore the use of blood profiling approaches to assess the immune status of an individual by using Primary Immune Deficiencies as a proof of principle.

Hypothesis

Experimental Design

A total of 22 blood samples were collected from three groups of subjects: 4 patients with complete MYD88 deficiency, 4 patients with complete IRAK4 deficiency, and 14 healthy donors. Mutations from IRAK4 and MYD88 patients are loss-of-function. The subjects were recruited in two different batches: first set includes 4 healthy controls, MYD88 P1 and P2, IRAK4 P1 and P2 patients. Second set includes 10 different healthy controls, MYD88 P3 and P4 and IRAK4 P3 and P4. Healthy individuals are considered so based on past medical history (no recurrent infections). They were also healthy at the time of sampling. From each subject, peripheral blood was drawn into sodium heparin vacuum tubes on clinic site (Necker Hospital, Paris). Right away, 500µl whole blood was diluted with the same amount of RPMI before adding different stimulus. Diluted whole blood was activated with a wide range of ligands (16 conditions total, see below) in a volume of 50 µl, for 2 hours along with 10µg/ml of polymyxin B to clear LPS contamination in reagents. Whole blood was lysated with Tempus solution (from Tempus tubes, Applied Biosystems) to stabilize the RNA at 1:3 ratio after restimulation. The lysates were kept at -80ºC temperature until mRNA extraction. A total of 365 samples were analyzed. Choice of agonists: PAM3 (TLR1/2) and PAM2 (TLR2/6), 100 ng/ml final concentration, Invivogen®; poly(I:C) 25 µg/ml final concentration, Invivogen® (TLR3); LPS 100 ng/ml final concentration, Sigma® (TLR4); Flagellin 100 ng/ml final concentration, Invivogen® (TLR5); 3M13 (TLR7) and 3M2 (TLR8), 3µg/ml final concentration, 3M pharmaceuticals®; R848 3µg/ml final concentration, Invivogen® (TLR7/8); CpG-D19 and CPG C (TLR9), 3µg/ml final concentration, from collaborator. Agonists for IL-1R: IL-1b 20ng/ml final concentration, IL18 and IL33 50ng/ml final concentration, from RaD systems®; agonist for TNFa receptor, TNFa, 20ng/ml final concentration, from RaD systems®; PMA 25 ng/ml + Ionomycin 1µg/ml final concentration, Sigma®; heat killed whole bacteria: 3 pneumococcal strains (R6, 5.106 partical/ml final concentration; R8450 108 partical/ml final concentration; R11470, 5.106 partical/ml final concentration, heat killed at 65°C for 15 min) from collaborator, and Staphylococcus aureus (SAC), 107 partical/ml final concentration, from Invivogen®. No stimulation serves as negative control and PMA/ionomycin stimulation as positive control.

Experimental Variables

Controls

Methods

Additional Information

Expected Samples:

72 Samples Loaded: 72

Species

Human (Homo sapiens)

Sample Source

Whole Blood

Disease

Primary Immunodeficiency

Treatment Protocol

Growth Protocol

Extraction Protocol

Storage Conditions

Sample Set Spreadsheet

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Samples Preview

Sample ID	Gender	Illness	!Sample_title	cell population	geographic
GSM632318	F	MYD88 patient	MYD88 P4_Flagellin	Whole blood	Turkish
GSM632319	F	MYD88 patient	MYD88 P3_IL-1b	Whole blood	Traveller (Gypsies)
GSM632322	F	MYD88 patient	MYD88 P3__3M2	Whole blood	Traveller (Gypsies)
GSM632328	F	MYD88 patient	MYD88 P4_TNFa	Whole blood	Turkish
GSM632331	F	MYD88 patient	MYD88 P4_No stimulation	Whole blood	Turkish

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Sample ID	Gender	Illness	!Sample Title	Cell population	Geographic	In vitro stimulation blood volume per condition	Age at enrollment	In vitro stimulation condition	Race	In vitro stimulation incubation time
GSM632318	F	MYD88 patient	MYD88 P4_Flagellin	Whole blood	Turkish	1 ml	5	Flagellin	White	2h
GSM632319	F	MYD88 patient	MYD88 P3_IL-1b	Whole blood	Traveller (Gypsies)	1 ml	2	IL1b	White	2h
GSM632322	F	MYD88 patient	MYD88 P3__3M2	Whole blood	Traveller (Gypsies)	1 ml	2	3M2	White	2h
GSM632328	F	MYD88 patient	MYD88 P4_TNFa	Whole blood	Turkish	1 ml	5	TNFa	White	2h
GSM632331	F	MYD88 patient	MYD88 P4_No stimulation	Whole blood	Turkish	1 ml	5	No stimulation	White	2h
GSM632332	F	MYD88 patient	MYD88 P3_No stimulation	Whole blood	Traveller (Gypsies)	1 ml	2	No stimulation	White	2h
GSM632333	F	MYD88 patient	MYD88 P3_SAC	Whole blood	Traveller (Gypsies)	1 ml	2	SAC	White	2h
GSM632335	F	MYD88 patient	MYD88 P4_IL-1b	Whole blood	Turkish	1 ml	5	IL1b	White	2h
GSM632338	F	MYD88 patient	MYD88 P3_TNFa	Whole blood	Traveller (Gypsies)	1 ml	2	TNFa	White	2h
GSM632339	F	MYD88 patient	MYD88 P4_SAC	Whole blood	Turkish	1 ml	5	SAC	White	2h
GSM632340	F	MYD88 patient	MYD88 P4_CpG-D19	Whole blood	Turkish	1 ml	5	CpGD19	White	2h
GSM632344	F	MYD88 patient	MYD88 P3_PMAIono	Whole blood	Traveller (Gypsies)	1 ml	2	PMAIono	White	2h
GSM632346	F	MYD88 patient	MYD88 P3_R11470	Whole blood	Traveller (Gypsies)	1 ml	2	R11470	White	2h
GSM632348	F	MYD88 patient	MYD88 P3_Flagellin	Whole blood	Traveller (Gypsies)	1 ml	2	Flagellin	White	2h
GSM632351	F	MYD88 patient	MYD88 P4_CpG C	Whole blood	Turkish	1 ml	5	CpG C	White	2h
GSM632352	F	MYD88 patient	MYD88 P4_R11470	Whole blood	Turkish	1 ml	5	R11470	White	2h
GSM632406	F	MYD88 patient	MYD88 P3_polyIC	Whole blood	Traveller (Gypsies)	1 ml	2	PolyIC	White	2h
GSM632411	F	MYD88 patient	MYD88 P4_IL33	Whole blood	Turkish	1 ml	5	IL33	White	2h
GSM632413	F	MYD88 patient	MYD88 P4_3M13	Whole blood	Turkish	1 ml	5	3M13	White	2h
GSM632417	F	MYD88 patient	MYD88 P3_3M13	Whole blood	Traveller (Gypsies)	1 ml	2	3M13	White	2h
GSM632420	F	MYD88 patient	MYD88 P4_IL18	Whole blood	Turkish	1 ml	5	IL18	White	2h
GSM632422	F	MYD88 patient	MYD88 P3_R8450	Whole blood	Traveller (Gypsies)	1 ml	2	R8450	White	2h
GSM632425	F	MYD88 patient	MYD88 P4_R848	Whole blood	Turkish	1 ml	5	R848	White	2h
GSM632427	F	MYD88 patient	MYD88 P4_LPS	Whole blood	Turkish	1 ml	5	LPS	White	2h
GSM632436	F	MYD88 patient	MYD88 P3_CpG-D19	Whole blood	Traveller (Gypsies)	1 ml	2	CpGD19	White	2h
GSM632442	F	MYD88 patient	MYD88 P3_PAM2	Whole blood	Traveller (Gypsies)	1 ml	2	PAM2	White	2h
GSM632443	F	MYD88 patient	MYD88 P4_polyIC	Whole blood	Turkish	1 ml	5	PolyIC	White	2h
GSM632447	F	MYD88 patient	MYD88 P4_PAM3	Whole blood	Turkish	1 ml	5	PAM3	White	2h
GSM632450	F	MYD88 patient	MYD88 P3_PAM3	Whole blood	Traveller (Gypsies)	1 ml	2	PAM3	White	2h
GSM632452	F	MYD88 patient	MYD88 P3_R6	Whole blood	Traveller (Gypsies)	1 ml	2	R6	White	2h
GSM632456	F	MYD88 patient	MYD88 P4_PAM2	Whole blood	Turkish	1 ml	5	PAM2	White	2h
GSM632457	F	MYD88 patient	MYD88 P3_R848	Whole blood	Traveller (Gypsies)	1 ml	2	R848	White	2h
GSM632460	F	MYD88 patient	MYD88 P4_R8450	Whole blood	Turkish	1 ml	5	R8450	White	2h
GSM632462	F	MYD88 patient	MYD88 P3_IL33	Whole blood	Traveller (Gypsies)	1 ml	2	IL33	White	2h
GSM632467	F	MYD88 patient	MYD88 P3_LPS	Whole blood	Traveller (Gypsies)	1 ml	2	LPS	White	2h
GSM632474	F	MYD88 patient	MYD88 P4_R6	Whole blood	Turkish	1 ml	5	R6	White	2h
GSM632476	F	MYD88 patient	MYD88 P4_PMAIono	Whole blood	Turkish	1 ml	5	PMAIono	White	2h
GSM632478	F	MYD88 patient	MYD88 P3_IL18	Whole blood	Traveller (Gypsies)	1 ml	2	IL18	White	2h
GSM632479	M	MYD88 patient	MYD88 P4__3M2	Whole blood	Turkish	1 ml	5	3M2	White	2h
GSM632480	F	MYD88 patient	MYD88 P3_CpG C	Whole blood	Traveller (Gypsies)	1 ml	2	CpG C	White	2h
GSM632486	M	MYD88 patient	MYD88 P2_CpG C	Whole blood	Portugal	1 ml	11	CpG C	White	2h
GSM632490	F	MYD88 patient	MYD88 P1_3M13	Whole blood	Portugal	1 ml	18	3M13	White	2h
GSM632492	F	MYD88 patient	MYD88 P1_IL18	Whole blood	Portugal	1 ml	18	IL18	White	2h
GSM632493	M	MYD88 patient	MYD88 P2_R848	Whole blood	Portugal	1 ml	11	R848	White	2h
GSM632494	F	MYD88 patient	MYD88 P1_TNFa	Whole blood	Portugal	1 ml	18	TNFa	White	2h
GSM632499	M	MYD88 patient	MYD88 P2_IL-1b	Whole blood	Portugal	1 ml	11	IL1b	White	2h
GSM632503	M	MYD88 patient	MYD88 P2_CpG-D19	Whole blood	Portugal	1 ml	11	CpGD19	White	2h
GSM632504	F	MYD88 patient	MYD88 P1_CpG-D19	Whole blood	Portugal	1 ml	18	CpGD19	White	2h
GSM632505	F	MYD88 patient	MYD88 P1__3M2	Whole blood	Portugal	1 ml	18	3M2	White	2h
GSM632513	M	MYD88 patient	MYD88 P2__3M2	Whole blood	Portugal	1 ml	11	3M2	White	2h
GSM632514	M	MYD88 patient	MYD88 P2_3M13	Whole blood	Portugal	1 ml	11	3M13	White	2h
GSM632515	M	MYD88 patient	MYD88 P2_PMAIono	Whole blood	Portugal	1 ml	11	PMAIono	White	2h
GSM632518	F	MYD88 patient	MYD88 P1_IL33	Whole blood	Portugal	1 ml	18	IL33	White	2h
GSM632529	M	MYD88 patient	MYD88 P2_LPS	Whole blood	Portugal	1 ml	11	LPS	White	2h
GSM632530	M	MYD88 patient	MYD88 P2_PAM3	Whole blood	Portugal	1 ml	11	PAM3	White	2h
GSM632541	M	MYD88 patient	MYD88 P2_PAM2	Whole blood	Portugal	1 ml	11	PAM2	White	2h
GSM632557	F	MYD88 patient	MYD88 P1_No stimulation	Whole blood	Portugal	1 ml	18	No stimulation	White	2h
GSM632558	F	MYD88 patient	MYD88 P1_R848	Whole blood	Portugal	1 ml	18	R848	White	2h
GSM632565	M	MYD88 patient	MYD88 P2_polyIC	Whole blood	Portugal	1 ml	11	PolyIC	White	2h
GSM632566	M	MYD88 patient	MYD88 P2_Flagellin	Whole blood	Portugal	1 ml	11	Flagellin	White	2h
GSM632567	F	MYD88 patient	MYD88 P1_CpG C	Whole blood	Portugal	1 ml	18	CpG C	White	2h
GSM632568	F	MYD88 patient	MYD88 P1_PMAIono	Whole blood	Portugal	1 ml	18	PMAIono	White	2h
GSM632571	F	MYD88 patient	MYD88 P1_IL-1b	Whole blood	Portugal	1 ml	18	IL1b	White	2h
GSM632577	F	MYD88 patient	MYD88 P1_PAM3	Whole blood	Portugal	1 ml	18	PAM3	White	2h
GSM632579	F	MYD88 patient	MYD88 P1_LPS	Whole blood	Portugal	1 ml	18	LPS	White	2h
GSM632581	F	MYD88 patient	MYD88 P1_Flagellin	Whole blood	Portugal	1 ml	18	Flagellin	White	2h
GSM632588	M	MYD88 patient	MYD88 P2_IL18	Whole blood	Portugal	1 ml	11	IL18	White	2h
GSM632589	F	MYD88 patient	MYD88 P1_PAM2	Whole blood	Portugal	1 ml	18	PAM2	White	2h
GSM632592	M	MYD88 patient	MYD88 P2_IL33	Whole blood	Portugal	1 ml	11	IL33	White	2h
GSM632601	F	MYD88 patient	MYD88 P1_polyIC	Whole blood	Portugal	1 ml	28	PolyIC	White	2h
GSM632604	M	MYD88 patient	MYD88 P2_No stimulation	Whole blood	Portugal	1 ml	11	No stimulation	White	2h
GSM632605	M	MYD88 patient	MYD88 P2_TNFa	Whole blood	Portugal	1 ml	11	TNFa	White	2h

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